Zinc Gluconate Induces Potentially Cancer Chemopreventive Activity in Barrett's Esophagus: A Phase 1 Pilot Study.

BACKGROUND: Chemopreventive effects of zinc for esophageal cancer have been well documented in animal models. This prospective study explores if a similar, potentially chemopreventive action can be seen in Barrett's esophagus (BE) in humans. AIMS: To determine if molecular evidence can be obtained potentially indicating zinc's chemopreventive action in Barrett's metaplasia. METHODS: Patients with a prior BE diagnosis were placed on oral zinc gluconate (14 days of 26.4 mg zinc BID) or a sodium gluconate placebo, prior to their surveillance endoscopy procedure. Biopsies of Barrett's mucosa were then obtained for miRNA and mRNA microarrays, or protein analyses. RESULTS: Zinc-induced mRNA changes were observed for a large number of transcripts. These included downregulation of transcripts encoding proinflammatory proteins (IL32, IL1ß, IL15, IL7R, IL2R, IL15R, IL3R), upregulation of anti-inflammatory mediators (IL1RA), downregulation of transcripts mediating epithelial-to-mesenchymal transition (EMT) (LIF, MYB, LYN, MTA1, SRC, SNAIL1, and TWIST1), and upregulation of transcripts that oppose EMT (BMP7, MTSS1, TRIB3, GRHL1). miRNA arrays showed significant upregulation of seven miRs with tumor suppressor activity (-125b-5P, -132-3P, -548z, -551a, -504, -518, and -34a-5P). Of proteins analyzed by Western blot, increased expression of the pro-apoptotic protein, BAX, and the tight junctional protein, CLAUDIN-7, along with decreased expression of BCL-2 and VEGF-R2 were noteworthy. CONCLUSIONS: When these mRNA, miRNA, and protein molecular data are considered collectively, a cancer chemopreventive action by zinc in Barrett's metaplasia may be possible for this precancerous esophageal tissue. These results and the extensive prior animal model studies argue for a future prospective clinical trial for this safe, easily-administered, and inexpensive micronutrient, that could determine if a chemopreventive action truly exists.

Release of sphingosine-1-phosphate from human platelets is dependent on thromboxane formation.

BACKGROUND: Platelets release the immune-modulating lipid sphingosine-1-phosphate (S1P). However, the mechanisms of platelet S1P secretion are not fully understood. OBJECTIVES: The present study investigates the function of thromboxane (TX) for platelet S1P secretion during platelet activation and the consequences for monocyte chemotaxis. METHODS: S1P was detected using thin-layer chromatography in [(3)H]sphingosine-labeled platelets and by mass spectrometry. Monocyte migration was measured in modified Boyden chamber chemotaxis assays. RESULTS: Release of S1P from platelets was stimulated with protease-activated receptor-1-activating peptide (PAR-1-AP, 100 µM). Acetylsalicylic acid (ASA) and two structurally unrelated reversible cyclooxygenase inhibitors diclofenac and ibuprofen suppressed S1P release. Oral ASA (500-mg single dose or 100 mg over 3 days) attenuated S1P release from platelets in healthy human volunteers ex vivo. This was paralleled by inhibition of TX formation. S1P release was increased by the TX receptor (TP) agonist U-46619, and inhibited by the TP antagonist ramatroban and by inhibitors of ABC-transport. Furthermore, thrombin-induced release of S1P was attenuated in platelets from TP-deficient mice. Supernatants from PAR-1-AP-stimulated human platelets increased the chemotactic capacity of human peripheral monocytes in a S1P-dependent manner via S1P receptors-1 and -3. These effects were inhibited by ASA-pretreatment of platelets. CONCLUSIONS: TX synthesis and TP activation mediate S1P release after thrombin receptor activation. Inhibition of this pathway may contribute to the anti-inflammatory actions of ASA, for example by affecting activity of monocytes at sites of vascular injury.

Sphingosine 1-phosphate: a regulator of arterial lesions.

Sphingosine-1 phosphate (S1P) is a bioactive sphingolipid that is critical in the development of blood vessels, and in the adult regulates vascular functions including vascular tone, endothelial integrity, and angiogenesis. Further, S1P may regulate arterial lesions in disease and after injury by controlling leukocyte recruitment and smooth muscle cell functions.

Lipid analysis of mitochondrial membranes from the yeast Pichia pastoris.

Here we describe for the first time isolation and biochemical characterization of highly purified mitochondrial inner and outer membranes from Pichia pastoris and systematic lipid analysis of submitochondrial fractions. Mitochondria of this yeast are best developed during growth on glycerol or sorbitol, but also on methanol or fatty acids. To obtain organelle membranes at high quality, methods of isolation and subfractionation of mitochondria originally developed for Saccharomyces cerevisiae were adapted and employed. A characteristic feature of the outer mitochondrial membrane of P. pastoris is the higher phospholipid to protein ratio and the lower ergosterol to phospholipid ratio compared to the inner membrane. Another marked difference between the two mitochondrial membranes is the phospholipid composition. Phosphatidylcholine and phosphatidylethanolamine are major phospholipids of both membranes, but the inner membrane is enriched in cardiolipin, whereas the outer membrane contains a high amount of phosphatidylinositol. The fatty acid composition of both mitochondrial membranes is similar. Variation of the carbon source, however, leads to marked changes of the fatty acid pattern both in total and mitochondrial membranes. In summary, our data are the first step to understand the P. pastoris lipidome which will be prerequisite to manipulate membrane components of this yeast for biotechnological purposes.

Dynamics of neutral lipid storage and mobilization in yeast.

We make use of the yeast Saccharomyces cerevisiae as a flexible experimental system to investigate coordinate pathways of neutral lipid synthesis, storage and mobilization with special emphasis on the role of different organelles in these processes. Recently, a number of new gene products involved in triacylglycerol (TAG) and steryl ester (STE) metabolism were identified in our laboratory and by other groups. STE are synthesized by the two STE synthases Are1p and Are2p, whereas TAG are formed mainly through the action of the two TAG synthases Dga1p and Lro1p with minor contributions of Are1p and Are2p. Once formed, TAG and STE are stored in so-called lipid particles. A dga1Deltalro1Deltaare1Deltaare2Delta quadruple mutant which lacks neutral lipid synthesis and is consequently devoid of lipid particles turned out to be a valuable tool for studying the physiological role of storage lipids and lipid particles. Mobilization of neutral lipid depots occurs through catalysis of TAG lipases and STE hydrolases. Three TAG lipases named Tgl3p, Tgl4p and Tgl5p, and three STE hydrolases named Tgl1p, Yeh1p and Yeh2p were recently identified at the molecular level. Although these hydrolases exhibit overlapping function within the enzyme families, they are specific for TAG and STE, respectively. With the exception of Dga1p, whose activity is partially localized to lipid particles, TAG and STE forming enzymes are restricted to the endoplasmic reticulum. TAG lipases and STE hydrolases are components of lipid particles with the exception of Yeh2p, which is plasma membrane located. Thus, neutral lipid metabolism is not only regulated at the enzyme level but also by the distribution of the components to organelles. The fact that neutral lipid homeostasis is linked to a number of cell biological processes confirms the important role of this class of lipids as cellular modulators or effectors.

The life cycle of neutral lipids: synthesis, storage and degradation.

Triacylglycerols (TAGs), steryl esters (SEs) and wax esters (WEs) form the group of neutral lipids. Whereas TAGs are present in all types of cell, the occurrence of SEs in prokaryotes is questionable, and the presence of WEs as storage molecules is restricted to plants and a few bacteria. Here, we summarize recent knowledge on the formation, storage and degradation of TAGs and SEs in various cell types. We describe the biochemical pathways involved in TAG and SE synthesis and discuss the subcellular compartmentation of these processes. Recently, several novel enzymes governing the metabolism of storage lipids have been identified and characterized. Regulatory aspects of neutral lipid storage are just beginning to be understood. Finally, we describe consequences of defects in neutral lipid metabolism. Since severe diseases like atherosclerosis, obesity and type 2 diabetes are caused by lipid accumulation, mechanisms underlying neutral lipid synthesis, depot formation and mobilization are of major interest for curing such diseases that are increasingly associated with modern civilization.

Formation and mobilization of neutral lipids in the yeast Saccharomyces cerevisiae.

Since energy storage is a basic metabolic process, the synthesis of neutral lipids occurs in all kingdoms of life. The yeast Saccharomyces cerevisiae, widely accepted as a model eukaryotic cell, contains two classes of neutral lipids, namely STEs (steryl esters) and TAGs (triacylglycerols). TAGs are synthesized through two pathways governed by the acyl-CoA diacylglycerol acyltransferase Dga1p and the phospholipid diacylglycerol acyltransferase Lro1p. STEs are formed by two STE synthases Are1p and Are2p, two enzymes with overlapping function, which also catalyse TAG formation, although to a minor extent. Neutral lipids are stored in the so-called lipid particles and can be utilized for membrane formation under conditions of lipid depletion. For this purpose, storage lipids have to be mobilized by TAG lipases and STE hydrolases. A TAG lipase named Tgl3p was identified as a major yeast TAG hydrolytic enzyme in lipid particles. Recently, a new family of hydrolases was detected which is required for STE mobilization in S. cerevisiae. These enzymes, named Yeh1p, Yeh2p and Tgl1p, are paralogues of the mammalian acid lipase family. The role of these proteins in biosynthesis and mobilization of TAG and STE, and the regulation of these processes will be discussed in this minireview.

Vasodilator-stimulated phosphoprotein is a substrate for protein kinase C.

Vasodilator-stimulated phosphoprotein (VASP), an actin binding protein localized to areas of focal contacts, is a substrate for the cyclic adenosine monophosphate/cyclic guanosine monophosphate (cAMP/cGMP)-dependent protein kinases (PKA, PKG). In this study, we show that serum stimulation of vascular smooth muscle cells (SMCs) induces VASP phosphorylation on Ser157, in a mechanism not dependent on PKA or PKG. We tested the possibility that protein kinase C (PKC), a regulator of cytoskeletal function, is involved. PKC inhibition or down-regulation prevented serum-induced phosphorylation of VASP at Ser157 in rat vascular SMCs. Additionally, recombinant PKCalpha directly phosphorylated Ser157 on VASP. In summary, our data support the hypothesis that PKC phosphorylates VASP and mediates serum-induced VASP regulation.

In yeast sterol biosynthesis the 3-keto reductase protein (Erg27p) is required for oxidosqualene cyclase (Erg7p) activity.

In Saccharomyces cerevisiae, the 3-keto reductase (Erg27p) encoded by ERG27 gene is one of the key enzymes involved in the C-4 demethylation of the sterol intermediate, 4,4-dimethylzymosterol. The oxidosqualene cyclase (Erg7p) encoded by the ERG7 gene converts oxidosqualene to lanosterol, the first cyclic component of sterol biosynthesis. In a previous study, we found that erg27 strains grown on cholesterol- or ergosterol-supplemented media did not accumulate lanosterol or 3-ketosterols but rather squalene, oxidosqualene, and dioxidosqualene intermediates normally observed in ERG7 (oxidosqualene cyclase) mutants. These results suggested a possible interaction between these two enzymes. In this study, we present evidence that Erg27p interacts with Erg7p, facilitating the association of Erg7p with lipid particles (LPs) and preventing digestion of Erg7p both in the endoplasmic reticulum (ER) and LPs. We demonstrate that Erg27p is required for oxidosqualene cyclase (Erg7p) activity in LPs, and that Erg27p co-immunoprecipitates with Erg7p in LPs but not in microsomal fractions. While Erg27p is essentially a component of the ER, it can also be detected in LPs. In erg27 strains, a truncated Erg7p mislocalizes to microsomes. Restoration of Erg7p enzyme activity and LPs localization was achieved in an erg27 strain transformed with a plasmid containing a wild-type ERG27 allele. We suggest that the physical interaction of Erg27p with Erg7p is an essential regulatory tool in yeast sterol biosynthesis.

Protein tyrosine phosphatase RQ is a phosphatidylinositol phosphatase that can regulate cell survival and proliferation.

Protein tyrosine phosphatase RQ (PTPRQ) was initially identified as a protein tyrosine phosphatase (PTPase)-like protein that is upregulated in a model of renal injury. Here we present evidence that, like PTEN, the biologically important enzymatic activity of PTPRQ is as a phosphatidylinositol phosphatase (PIPase). The PIPase specificity of PTPRQ is broader than that of PTEN and depends on different amino acid residues in the catalytic domain. In vitro, the recombinant catalytic domain of PTPRQ has low PTPase activity against tyrosine-phosphorylated peptide and protein substrates but can dephosphorylate a broad range of phosphatidylinositol phosphates, including phosphatidylinositol 3,4,5-trisphosphate and most phosphatidylinositol monophosphates and diphosphates. Phosphate can be hydrolyzed from the D3 and D5 positions in the inositol ring. PTPRQ does not have either of the basic amino acids in the catalytic domain that are important for the PIPase activity of PTEN or the sequence motifs that are characteristic of type II phosphatidylinositol 5-phosphatases. Instead, the PIPase activity depends on the WPE sequence present in the catalytic cleft of PTPRQ, and in the "inactive" D2 domains of many dual-domain PTPases, in place of the WPD motif present in standard active PTPases. Overexpression of PTPRQ in cultured cells inhibits proliferation and induces apoptosis. An E2171D mutation that retains or increases PTPase activity but eliminates PIPase activity, eliminates the inhibitory effects on proliferation and apoptosis. These results indicate that PTPRQ represents a subtype of the PTPases whose biological activities result from its PIPase activity rather than its PTPase activity.

Triacylglycerol biosynthesis in yeast.

Triacylglycerol (TAG) is the major storage component for fatty acids, and thus for energy, in eukaryotic cells. In this mini-review, we describe recent progress that has been made with the yeast Saccharomyces cerevisiae in understanding formation of TAG and its cell biological role. Formation of TAG involves the synthesis of phosphatidic acid (PA) and diacylglycerol (DAG), two key intermediates of lipid metabolism. De novo formation of PA in yeast as in other types of cells can occur either through the glycerol-3-phosphate- or dihydroxyacetone phosphate-pathways-each named after its respective precursor. PA, formed in two steps of acylation, is converted to DAG by phosphatidate phosphatase. Acylation of DAG to yield TAG is catalyzed mainly by the two yeast proteins Dga1p and Lro1p, which utilize acyl-CoA or phosphatidylcholine, respectively, as acyl donors. In addition, minor alternative routes of DAG acylation appear to exist. Endoplasmic reticulum and lipid particles (LP), the TAG storage compartment in yeast, are the major sites of TAG synthesis. The interplay of these organelles, formation of LP, and enzymatic properties of enzymes catalyzing the synthesis of PA, DAG, and TAG in yeast are discussed in this communication.

The roles of phosphatidylinositol 3-kinase and protein kinase Czeta for thrombopoietin-induced mitogen-activated protein kinase activation in primary murine megakaryocytes.

Thrombopoietin (TPO) stimulates a network of intracellular signaling pathways that displays extensive cross-talk. We have demonstrated previously that the ERK/mitogen-activated protein kinase pathway is important for TPO-induced endomitosis in primary megakaryocytes (MKs). One known pathway by which TPO induces ERK activation is through the association of Shc with the penultimate phosphotyrosine within the TPO receptor, Mpl. However, several investigators found that the membrane-proximal half of the cytoplasmic domain of Mpl is sufficient to activate ERK in vitro and support base-line megakaryopoiesis in vivo. Using BaF3 cells expressing a truncated Mpl (T69Mpl) as a tool to identify non-Shc/Ras-dependent signaling pathways, we describe here novel mechanisms of TPO-induced ERK activation mediated, in part, by phosphoinositide 3-kinase (PI3K). Similar to cells expressing full-length receptor, PI3K was activated by its incorporation into a complex with IRS2 or Gab2. Furthermore, the MEK-phosphorylating activity of protein kinase Czeta (PKCzeta) was also enhanced after TPO stimulation of T69Mpl, contributing to ERK activity. PKCzeta and PI3K also contribute to TPO-induced ERK activation in MKs, confirming their physiological relevance. Like in BaF3 cells, a TPO-induced signaling complex containing p85PI3K is detectable in MKs expressing T61Mpl and is probably responsible for PI3K activation. These data demonstrate a novel role of PI3K and PKCzeta in steady-state megakaryopoiesis.

Roles of phosphatidylethanolamine and of its several biosynthetic pathways in Saccharomyces cerevisiae.

Three different pathways lead to the synthesis of phosphatidylethanolamine (PtdEtn) in yeast, one of which is localized to the inner mitochondrial membrane. To study the contribution of each of these pathways, we constructed a series of deletion mutants in which different combinations of the pathways are blocked. Analysis of their growth phenotypes revealed that a minimal level of PtdEtn is essential for growth. On fermentable carbon sources such as glucose, endogenous ethanolaminephosphate provided by sphingolipid catabolism is sufficient to allow synthesis of the essential amount of PtdEtn through the cytidyldiphosphate (CDP)-ethanolamine pathway. On nonfermentable carbon sources, however, a higher level of PtdEtn is required for growth, and the amounts of PtdEtn produced through the CDP-ethanolamine pathway and by extramitochondrial phosphatidylserine decarboxylase 2 are not sufficient to maintain growth unless the action of the former pathway is enhanced by supplementing the growth medium with ethanolamine. Thus, in the absence of such supplementation, production of PtdEtn by mitochondrial phosphatidylserine decarboxylase 1 becomes essential. In psd1Delta strains or cho1Delta strains (defective in phosphatidylserine synthesis), which contain decreased amounts of PtdEtn, the growth rate on nonfermentable carbon sources correlates with the content of PtdEtn in mitochondria, suggesting that import of PtdEtn into this organelle becomes growth limiting. Although morphological and biochemical analysis revealed no obvious defects of PtdEtn-depleted mitochondria, the mutants exhibited an enhanced formation of respiration-deficient cells. Synthesis of glycosylphosphatidylinositol-anchored proteins is also impaired in PtdEtn-depleted cells, as demonstrated by delayed maturation of Gas1p. Carboxypeptidase Y and invertase, on the other hand, were processed with wild-type kinetics. Thus, PtdEtn depletion does not affect protein secretion in general, suggesting that high levels of nonbilayer-forming lipids such as PtdEtn are not essential for membrane vesicle fusion processes in vivo.

A subfraction of the yeast endoplasmic reticulum associates with the plasma membrane and has a high capacity to synthesize lipids.

Large parts of the endoplasmic reticulum of the yeast, Saccharomyces cerevisiae, are located close to intracellular organelles, i.e. mitochondria and the plasma membrane, as shown by fluorescence and electron microscopy. Here we report the isolation and characterization of the subfraction of the endoplasmic reticulum that is closely associated with the plasma membrane. This plasma membrane associated membrane (PAM) is characterized by its high capacity to synthesize phosphatidylserine and phosphatidylinositol. As such, PAM is reminiscent of MAM, a mitochondria associated membrane fraction of the yeast [Gaigg, B., Simbeni, R., Hrastnik, C., Paltauf, F. & Daum, G. (1995) Biochim. Biophys. Acta 1234, 214-220], although the specific activity of phosphatidylserine synthase and phosphatidylinositol synthase in PAM exceeds several-fold the activity in MAM and also in the bulk endoplasmic reticulum. In addition, several enzymes involved in ergosterol biosynthesis, namely squalene synthase (Erg9p), squalene epoxidase (Erg1p) and steroldelta24-methyltransferase (Erg6p), are highly enriched in PAM. A possible role of PAM in the supply of lipids to the plasma membrane is discussed.

Arsenite inhibits Ras-dependent activation of ERK but activates ERK in the presence of oncogenic Ras in baboon vascular smooth muscle cells.

Exposure to arsenical compounds enhances the risk of atherosclerosis. The reason is unknown but it might be because an effect of arsenite (As3+) on plaque smooth muscle cells (SMCs) activation of extracellular signal-regulated kinase (ERK), a crucial mediator of SMC function. We found that arsenite inhibits the activation of ERK by platelet-derived growth factor-BB (PDGF-BB). This inhibitory effect depends on the time of arsenite exposure, is reversible, and is attenuated by preincubation of SMCs with the antioxidant N-acetyl-cysteine. These observations are consistent with the assumption that oxidative stress is involved. The blockade of ERK by arsenite may be mediated by an inhibition of Ras as arsenite prevents GTP-loading of Ras in response to PDGF-BB. Moreover, the Ras blockade by arsenite is not specific for PDGF-BB because it was also observed following stimulation of SMCs with EGF. To address the role of Ras, we expressed constitutively active, GTP-bound Ha-Ras (V12Ras). Unexpectedly, in V12Ras expressing-SMCs, arsenite stimulates ERK, but still decreases ERK activity in the presence of PDGF-BB. Our data suggest that arsenite inhibits the Ras/ERK pathway in SMCs, and that arsenite may activate ERK in Ras-transformed cells by mechanisms different from those employed by growth factors.

Regulation of the Saccharomyces cerevisiae DPP1-encoded diacylglycerol pyrophosphate phosphatase by zinc.

The DPP1 gene, encoding diacylglycerol pyrophosphate (DGPP) phosphatase from Saccharomyces cerevisiae, has recently been identified as a zinc-regulated gene, and it contains a putative zinc-responsive element (UAS(ZRE)) in its promoter. In this work we examined the hypothesis that expression of DGPP phosphatase was regulated by zinc availability. The deprivation of zinc from the growth medium resulted in a time- and dose-dependent induction of beta-galactosidase activity driven by a P(DPP1)-lacZ reporter gene. This regulation was dependent on the UAS(ZRE) in the DPP1 promoter and was mediated by the Zap1p transcriptional activator. Induction of the DGPP phosphatase protein and activity by zinc deprivation was demonstrated by immunoblot analysis and measurement of the dephosphorylation of DGPP. The regulation pattern of DGPP phosphatase in mutants defective in plasma membrane (Zrt1p and Zrt2p) and vacuolar membrane (Zrt3p) zinc transporters indicated that enzyme expression was sensitive to the cytoplasmic levels of zinc. DGPP phosphatase activity was inhibited by zinc by a mechanism that involved formation of DGPP-zinc complexes. Studies with well characterized subcellular fractions and by indirect immunofluorescence microscopy revealed that the DGPP phosphatase enzyme was localized to the vacuolar membrane.

EGFR transactivation in the regulation of SMC function.

Vascular smooth muscle cells (SMCs) are the principal cellular component of the normal artery and intimal lesions that develop in response to arterial injury. Several growth factors and their receptors participate in SMC activation, including the tyrosine kinase receptors for platelet-derived growth factor (PDGF) and basic fibroblast growth factor as well as the G-protein-coupled receptors (GPCRs) for thrombin and angiotensin II. During the last couple of years, it has become evident that GPCRs transactivate receptor tyrosine kinases, particularly the epidermal growth factor receptor (EGFR). The EGFR is not well characterized in terms of its role in vascular biology, but recent findings indicate that GPCRs induce EGFR transactivation in cultured vascular SMCs, perhaps by intracellular and extracellular pathways. Studies from our laboratory as well as two other groups have demonstrated that EGFR transactivation by different GPCR agonists and in different cell types, including SMCs, is mediated by heparin-binding EGF-like growth factor (HB-EGF). HB-EGF-dependent EGFR activation is blocked by heparin, a growth inhibitor of SMCs in vitro and in vivo. These data suggest that the EGFR may be important in the regulation of SMC function. The complexity of the GPCR-EGFR crosstalk, involving several different cell surface molecules and an inside-out signaling step, may provide novel targets for the control of SMC growth and intimal hyperplasia in the arterial injury response.

Screening for drug and alcohol abuse among older adults using a modified version of the CAGE.

This study examined the sensitivity, specificity, and receiver operating characteristics (ROC) curves of a modified version of the CAGE, a screening measure used in the detection of older alcohol- and drug-abusing individuals. In a retrospective review of clinical records of 976 patients screened by a geriatric substance abuse program, the authors examined patients' responses on a modified version of the CAGE that included queries regarding drug use. The CAGE was administered to individuals age 50 or over draw from three diagnostic groups: alcohol abuse/dependence, drug abuse/dependence, and normal controls. Analysis of variance and discriminant function analyses revealed that the modified CAGE was able to discriminate both alcohol and drug abusers from controls. Analyses examining test sensitivity, specificity, and ROC curves revealed the CAGE to demonstrate excellent sensitivity but poor specificity. Omitting the "cut down'' item from the CAGE significantly improved specificity with only a modest drop in sensitivity. Given the ease of administration and sensitivity to both alcohol and drug abuse, these data suggest that the modified CAGE is well suited as a screening instrument for geriatric drug and alcohol abuse.

Intracellular lipid particles of eukaryotic cells.

In this review article we describe characterization of intracellular lipid particles of three different eukaryotic species, namely mammalian cells, plants and yeast. Lipid particles of all types of cells share a general structure. A hydrophobic core of neutral lipids is surrounded by a membrane monolayer of phospholipids which contains a minor amount of proteins. Whereas lipid particles from mammalian cells and plants harbor specific classes of polypeptides, mainly perilipins and oleosins, respectively, yeast lipid particles contain a more complex set of enzymes which are involved in lipid biosynthesis. Function of lipid particles as storage compartment and metabolic organelle, and their interaction with other subcellular fractions are discussed. Furthermore, models for the biogenesis of lipid particles are presented and compared among the different species.

Heparin blockade of thrombin-induced smooth muscle cell migration involves inhibition of epidermal growth factor (EGF) receptor transactivation by heparin-binding EGF-like growth factor.

Agonists of G protein-coupled receptors, such as thrombin, act in part by transactivating the epidermal growth factor (EGF) receptor (EGFR). Although at first a ligand-independent mechanism for EGFR transactivation was postulated, it has recently been shown that this transactivation by various G protein-coupled receptor agonists can involve heparin-binding EGF-like growth factor (HB-EGF). Because thrombin stimulation of vascular smooth muscle cell migration is blocked by heparin and because heparin can displace HB-EGF, we investigated the possibility that thrombin stimulation of smooth muscle cells (SMCs) depends on EGFR activation by HB-EGF. In rat SMCs, EGFR phosphorylation and extracellular signal-regulated kinase (ERK) activation in response to thrombin are inhibited not only by the EGFR inhibitor AG1478 and by EGFR blocking antibody but also by heparin and by neutralizing HB-EGF antibody. HB-EGF-dependent signaling induced by thrombin is inhibited by batimastat, which suggests a requirement for pro-HB-EGF shedding by a metalloproteinase. We further demonstrate that this novel pathway is required for the migration of rat and baboon SMCs in response to thrombin. We conclude from these data that the inhibitory effect of heparin on SMC migration induced by thrombin relies, at least in part, on a blockade of HB-EGF-mediated EGFR transactivation.